Project Summary Biological macromolecules exhibit an amazing degree of conformational heterogeneity as required for their various functions. The importance of this heterogeneity is becoming more evident as different biological functions associated with various conformational states of individual biological molecules are identified. To investigate the conformational properties of macromolecules and facilitate the use of the information in drug development, our laboratory has focused on a comprehensive research program that optimizes and extends empirical force fields for biological and drug-like molecules, develops novel conformational and solute sampling methods and applies those tools in collaborative studies on systems of therapeutic relevance. In the proposed studies we will further optimize and extend both the additive (fixed-charge) CHARMM and polarizable classical Drude oscillator force fields. Work on the Drude force field will involve extensions to cover the full range of biological macromolecules and organic, drug-like molecules, continue to improve the overall accuracy of the model, extend the model to more accurately treat ligated metals via the inclusion of local charge transfer effects and implement improved methods for the treatment of van der Waals interactions. Sampling methods development will extend the Hamiltonian Replica Exchange approach to enhance sampling in oligonucleotides and polysaccharides including improved sampling of specific degrees of freedom associated with high-energy barriers using biasing potentials. The solute sampling method developed in our laboratory based on the oscillating ?ex Grand-Canonical Monte Carlo/Molecular Dynamics method will be extended to more accurately sample the distribution of osmolytes and ions, including Mg+2, around macromolecules and allow the approach to be used with the polarizable Drude force field. In combination, the conformational and solute sampling approaches represent powerful methods that will allow for theoretical investigations of the interplay between environment and macromolecular conformational heterogeneity. The developed tools will be applied in studies on nucleic acids investigating the ionic atmosphere of DNA, exploiting solvachromatic shifts determined using QM/MM methods, the impact of Mg+2 on the conformational heterogeneity of RNA, including on riboswtiches and small regulatory RNAs in bacterial pathogens, and the catalytic and base specificity mechanisms of DNA glycosylases important for base excision repair. In the area of polysaccharides, the conformational heterogeneity of glycans acting as antigens for vaccines targeting antibiotic resistant bacteria and for use in cancer immunotherapy will be investigated. Specific disease states to be targeted include antibiotic resistant infections associated with Klebsiella Pneumonia and Pseudomonas Aeruginsa and cancers accessible to immunotherapy treatment. In addition, these collaborative efforts will further validate the developed force fields and methods, tools that are available to and widely used by the scientific community.